Anti p mtor antibody

The following primary antibodies were used in this study: anti-PIWIL1 antibody (ab12337; Abcam, Cambridge, UK), anti-p-mTOR antibody (ab109268; Abcam), anti-LC3 I/II (#2775S; Cell Signaling Technology, Danvers, MA, USA), anti-P62/SQSTM1 (#8025S; Cell Signaling Technology), anti-Parkin (#4211; Cell Signaling Technology), anti-phosphor-NAK/TBK (ab109272; Abcam), anti-NAK/TBK (A5497; Bimake), anti-optineurin (ab213556; Abcam), anti-β-actin (#4970; Cell Signaling Technology), anti-Akt (#4691; Cell Signaling Technology), anti-phospho-Akt (Ser473) (#9271; Cell Signaling Technology), anti-Ki67 (#4685; Cell Signaling Technology), anti-Bcl-2 (A5010; Bimake), anti-OCT4 (60242-1-Ig; Proteintech), and anti-Nanog (#4903T; Cell Signaling Technology). Goat anti-mouse and goat anti-rabbit antibodies (Biosciences) were used as the secondary antibodies for western blotting. Dexamethasone and doxorubicin were purchased from Sigma (St. Louis, MO, USA), and bortezomib was purchased from Selleck Chemicals. 3-Methyladenine (3-MA) and the mitophagy inhibitor cyclosporin A (CsA) were purchased from MedChemExpress (NJ, USA). All were dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C for up to 6 months. For all cell-based experiments, drugs were diluted at least by 1:1000 to ensure that the final DMSO concentration was lower than 0.1%.

Recombinant rat Netrin-1 was purchased from Creative Biomart (Shirley, NY, USA). Anti-LC3B, anti-Beclin-1, anti-NeuN, anti-mTOR, and anti-p-mTOR antibody as well as goat anti-rabbit and goat anti-mouse-IgG HRP were purchased from Abcam (Cambridge, MA, USA). Anti-AMPK, anti-p-AMPK, anti-p-P70S6K, anti-P70S6K, and anti-caspase-3 antibody were obtained from Cell Signaling Technology (Danvers, MA, USA). Alexa Fluor^® 568 and Alexa Fluor^® 488 were acquired from Life Technology (Carlsbad, CA, USA). Anti-β-tubulin antibody was provided by TransGen Biotech (Beijing, China). Dorsomorphin (Compound C), an AMPK inhibitor, was supplied by Selleck Chemicals LLC (Houston, TX, USA). An enhanced chemiluminescence (ECL) kit was obtained from Beyotime Institute of Biotechnology (Nanjing, Jiangsu, China). In situ Cell Death Detection Kit was purchased from Roche (Mannheim, Germany). All of the other reagents were acquired from Sigma–Aldrich (St. Louis, MO, USA) unless otherwise specified.

Paraffin-embedded lung sections were deparaffinized with xylene and hydrated using graded alcohols. The mTORC1 activity was assessed using anti-p-mTOR antibody (1:800, Abcam, Cambridge, UK) and anti-p-S6RP antibody (1:200, Cell Signaling Technology, MA). Macrophage infiltration was detected using anti-CD68 antibody (1:200, Abcam, Cambridge, UK) [78 (link), 79 (link)]. The antibody was then detected using a standard streptavidin-biotin detection system (Beijing Zhongshan Biotechnology Co., Ltd., Beijing, China) according to the manufacturer’s instructions.